Polyamines regulate gene expression by stimulating translation of histone acetyltransferase mRNAs

Abstract

Polyamines regulate gene expression in Escherichia coli by translationally stimulating mRNAs encoding global transcription factors. In this study, we focused on histone acetylation, one of the mechanisms of epigenetic regulation of gene expression, to attempt to clarify the role of polyamines in the regulation of gene expression in eukaryotes. We found that activities of histone acetyltransferases in both the nucleus and cytoplasm decreased significantly in polyamine-reduced mouse mammary carcinoma FM3A cells. Although protein levels of histones H3 and H4 did not change in control and polyamine-reduced cells, acetylation of histones H3 and H4 was greatly decreased in the polyamine-reduced cells. Next, we used control and polyamine-reduced cells to identify histone acetyltransferases whose synthesis is stimulated by polyamines. We found that polyamines stimulate the translation of histone acetyltransferases GCN5 and HAT1. Accordingly, GCN5- and HAT1-catalyzed acetylation of specific lysine residues on histones H3 and H4 was stimulated by polyamines. Consistent with these findings, transcription of genes required for cell proliferation was enhanced by polyamines. These results indicate that polyamines regulate gene expression by enhancing the expression of the histone acetyltransferases GCN5 and HAT1 at the level of translation. Mechanistically, polyamines enhanced the interaction of microRNA-7648-5p (miR-7648-5p) with the 5'-UTR of GCN5 mRNA, resulting in stimulation of translation due to the destabilization of the double-stranded RNA (dsRNA) between the 5'-UTR and the ORF of GCN5 mRNA. Because HAT1 mRNA has a short 5'-UTR, polyamines may enhance initiation complex formation directly on this mRNA.

Keywords: gene expression; histone acetylase; histone acetyltransferase; histone acetyltransferase 1 (HAT1); histone acetyltransferase GCN5 (GCN5); histone modification; miR-7648-5p; polyamine; polyamine modulon; spermidine; translation regulation; translational regulation.

Figure 1.

Stimulation of acetylation of histones H3 and H4 by polyamines. FM3A cells were cultured in the presence and absence of 50 μm DFMO for 72 h. Nucleus and cytoplasm fractions were prepared, and activity of histone acetyltransferases (HATs) was measured as described under “Experimental Procedures.” (A) Activity of histone acetyltransferases. (B) Levels of histones and their acetylation. * p < 0.05; ** p < 0.01.

Figure 2.

Western blotting of histone acetyltransferases (A), and the levels of mRNA of Gcn5 and Hat1 (B). (A) Protein levels of 13 histone acetyltransferases and of β-actin as a control were determined by Western blotting as described under “Experimental Procedures” using 20 μg of cell lysate, and the ratio of control/DFMO-treated cells was shown. *** p < 0.001. (B) mRNA levels of Gcn5, Hat1, and β-actin were determined as described under “Experimental Procedures.”

Figure 3.

Comparison of polyamine stimulation of histone acetylation at each lysine residue in histones H3 and H4. (A) Acetylation sites of histones H3 and H4 by Gcn5 and Hat1 (17). (B) Level of acetylation of H3 and H4. Histones were isolated from control and DFMO-treated, polyamine-reduced FM3A cells and analyzed by Western blotting using site-specific acetyl lysine antibodies as described under “Experimental Procedures.” * p < 0.05; ** p < 0.01.

Figure 4.

Stimulation of the synthesis of several mRNAs by polyamines through stimulation of acetylation of lysine residues in histones H3 and H4. (A) mRNA levels of cell growth-related genes, K_i-67, Lyar, Pcna, and MCM-2 and of PRL30 as a control. mRNA was isolated from control and DFMO-treated FM3A cells, and cDNA was synthesized and analyzed by quantitative real-time PCR as described under “Experimental Procedures.” * p < 0.05; ** p < 0.01. (B) Analysis of active promoter regions. The ChIP assay was carried out using anti-site-specific acetyl lysine of histones H3 and H4, and promoter regions of cell growth related genes, Ki-67, Lyar, Pcna, and MCM-2, and PRL30 as a control were amplified. Experiments were repeated twice, and the mean ratio (control/DFMO-treated) is shown. More than 1.5 is shown in red.

Figure 5.

Identification of polyamine acting sites on Gcn5 and Hat1 mRNAs. WT and 5′-UTR-deleted Gcn5-EGFP and Hat1-EGFP fusion plasmids were transfected into NIH3T3 cells and cultured in the presence and absence of 500 μm DFMO for 72 h. Protein levels were analyzed by Western blotting using anti-EGFP antibody (Clontech). ** p < 0.01; *** p < 0.001.

Figure 6.

Involvement of miR-7648-5p on the polyamine stimulation of Gcn5 synthesis. (A) Initiation region of Gcn5 mRNA (–63 to +70) and miR-7648-5p sequences are shown. (B) WT and 5′-UTR mutated Gcn5-EGFP constructs. (C, D) Expression levels of WT and mutated Gcn5-EGFP fusion proteins were analyzed by fluorescence microscopy and by Western blotting using lysates of NIH3T3 cells cultured in the presence and absence of 500 μm DFMO. *** p < 0.001.

Figure 7.

Effect of miR-7648-5p on polyamine stimulation of Gcn5 synthesis. (A) MicroRNA expression plasmid pCMV-MIR7648 and its vector were transfected into NIH3T3, and the effect of polyamines was examined using DFMO-treated and untreated cells. (B) MicroRNA expression plasmid pCMV-MIR7648 and its vector were cotransfected with pGcn5-EGFP into NIH3T3 cells. Protein levels of Gcn5 and Gcn5-EGFP were determined by Western blotting as described under “Experimental Procedures” using 20 μg of cell lysate, and the ratio of control/DFMO-treated cells was shown. *** p < 0.001.

Figure 8.

CD analysis of the complex between the 5′-UTR of Gcn5 mRNA and miR-7648-5p in the presence and absence of spermine. (A) Structures of Gcn5-miR-7648-5p fusion RNA. (B) CD spectra were recorded as described under “Experimental Procedures.” (C) Concentration-dependent shifts induced by Mg²⁺ (■), spermine (●), and spermine with 1 mm Mg²⁺ (□) at 37 °C in magnitude at 208 nm are shown. Values are means ± S.E. of triplicate determinations. The K_d values were determined according to the double reciprocal equation plot.